All referenced patents and applications are incorporated herein by reference in their entirety. Furthermore, where a definition or use of a term in a reference, which is incorporated by reference herein is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply.
Small scale bioreactors are intended to reduce the effort required to perform complicated fermentation and cell culture experiments, such as continuous cultures, and to provide the possibility of conducting these experiments in parallel. Continuous culture experiments, described for example in US2011/0053806A1, require the aseptic introduction of large volumes of fluid to the microfluidic device, typically many times the internal device volume. This requires an aseptic interconnection between at least one large volume aseptic reservoir and the aseptic microfluidic device. For parallel experiments, performing many of these aseptic connections efficiently is important. Conventional means to perform aseptic connections include the use of septa that block openings to channels in the microfluidic device, for example as in US2008/0241909A1 and puncturing the septa with a sterile needle connected to a source of sterile fluid, or making manual tubing connections between the fluid sources and the microfluidic device in a sterile environment. These methods require manual dexterity and are inconvenient when setting up many experiments.
There is also the requirement to supply a driving force to introduce the fluid into the device. This is typically done by supplying the fluid at a higher pressure than the fluid pressure in the device to generate a pressure driven flow. Under these conditions, with the fluid source at a pressure higher than the device, there is a risk of unintentional introduction of fluid to the area around the device should it be disconnected before equalizing the fluid pressure to atmospheric pressure. The unintentionally released fluid could damage sensitive equipment or contaminate clean surfaces.
An additional consideration for small scale bioreactors is loss of fluid due to evaporation. This is due to the large surface area to volume ratio of typical microfluidic devices. Minimizing fluid loss due to evaporation is important in order to minimize changes in the concentration of nutrients. Minimization of evaporation is particularly challenging because of the relatively high rate of gas exchange required to provide mixing and oxygenation to growing organisms. One approach to minimizing evaporation is to ensure the relative humidity of the gas that is exchanged with the bioreactor is near 100%. However, maintaining a high relative humidity of the gas increases the risk of condensation. Condensation of water has the negative effects of clogging channels designed for gas flow with liquid, and also interfering with optical or electrical measurements. To reduce or prevent condensation, any surfaces in contact with the humidified gas are set to temperatures higher than the condensation temperature of the humidified gas or the gas is dehumidified before reaching the surfaces of interest as in US5458008. However, since it is necessary to provide humidified gas to the devices performing liquid and gas reactions, dehumidifying the gas to prevent condensation before reaching the device is detrimental to system operation since it will not prevent fluid loss. Yet another method employed by US 2011/0076759 A1 is to implement a condenser at the output of the device to prevent evaporated liquid from leaving the device. While this method works in larger systems, smaller systems typically cannot afford to have cold regions close to temperature controlled or heated regions and systems relying on membranes for gas transfer have no method of returning condensed liquid to the device for reuse.
Thus, there is a need for an apparatus and methods to operate microfluidic bioreactors that provides convenient aseptic connections between microfluidic devices and large volume fluid reservoirs; mechanisms to reduce the chance of unintentional introduction of fluid around the device; and minimization of evaporation while at the same time avoiding condensation. In addition, such apparatus and methods should be efficient enough to use for many microfluidic bioreactors operated in parallel.